Product Ref.

3222
Product Desc. Phospholipid-8Pro-A
Manual Rev. No. 003 : 2015-12-09

Instruction Manual

Table of Contents

1 Intended Use .................................................................................................................. 1

2 Clinical Application and Principle of the Assay ................................................................ 1
3 Kit Contents .................................................................................................................... 2
4 Storage and Shelf Life .................................................................................................... 2
5 Precautions of Use.......................................................................................................... 3
6 Sample Collection, Handling and Storage ....................................................................... 4
7 Assay Procedure ............................................................................................................ 4
8 Qualitative Interpretation ................................................................................................. 7
9 Technical Data ................................................................................................................ 8
10 Performance Data ........................................................................................................... 8
11 Literature ........................................................................................................................ 9

AESKU.DIAGNOSTICS GmbH & Co. KG

Mikroforum Ring 2
55234 Wendelsheim, Germany
Tel: +49-6734-9622-0
Fax: +49-6734-9622-2222
Info@aesku.com
www.aesku.com
 Product Ref. 3222
Product Desc. Phospholipid-8Pro-A
Manual Rev. No. 003 : 2015-12-09

1 Intended Use
AESKULISA Phospholipid-8Pro-A is a solid phase enzyme immunoassay for the separate
qualitative detection of IgA antibodies against phospholipids in human serum. The assay
employs highly purified human ß2-Glycoprotein I, Cardiolipin + ß2-Glycoprotein I, Cardiolipin
and Phosphatidylcholin, -ethanolamin, -inositol, -serine and Sphingomyelin.
The assay is an aid in the diagnosis and risk estimation of thrombosis in patients with
systemic lupus erythematosus.

2 Clinical Application and Principle of the Assay

Antibodies against phospholipids, components of the biological membranes, are specific for
phospholipids such as Cardiolipin, Phosphatidyl -inositol, -ethanolamine, -choline,
Sphingomyelin and phosphatidic acid.
Anti-phospholipid antibodies are frequently found in sera of patients with systemic lupus
erythematosus (SLE) and related diseases. The occurrence of anti-phospholipid antibodies
in patients with SLE and related diseases is typical for a secondary anti-phospholipid
syndrome (APS). In contrast, anti-phospholipid antibodies in patients with no other
autoimmune diseases characterize the primary APS.
Many studies have shown a correlation between these autoantibodies and an enhanced
incidence of thrombosis, thrombocytopenia and habitual abortions (as a consequence of
placental infarct). The exact mechanisms by which pathogenic anti-phospholipid antibodies
induce thrombosis is not yet revealed fully.

Principle of the test

Serum samples diluted 1:101 are incubated in the microplates coated with the specific
antigen. Patient´s antibodies, if present in the specimen, bind to the antigen. The
unbound fraction is washed off in the following step. Afterwards anti-human
immunoglobulins conjugated to horseradish peroxidase (conjugate) are incubated and
react with the antigen-antibody complex of the samples in the microplates. Unbound
conjugate is washed off in the following step. Addition of TMB-substrate generates an
enzymatic colorimetric (blue) reaction, which is stopped by diluted acid (color changes
to yellow). The intensity of color formation from the chromogen is a function of the
amount of conjugate bound to the antigen-antibody complex and this is proportional to
the initial concentration of the respective antibodies in the patient sample.

Page 1 of 9
 Product Ref. 3222
Product Desc. Phospholipid-8Pro-A
Manual Rev. No. 003 : 2015-12-09

5 Precautions of Use
5.1 Health hazard data
THIS PRODUCT IS FOR IN VITRO DIAGNOSTIC USE ONLY. Thus, only staff trained and
specially advised in methods of in vitro diagnostics may perform the kit. Although this product
is not considered particularly toxic or dangerous in conditions of the intended use, refer to the
following for maximum safety:
Recommendations and precautions
This kit contains potentially hazardous components. Though kit reagents are not classified
being irritant to eyes and skin we recommend to avoid contact with eyes and skin and wear
disposable gloves.
WARNING ! Calibrators, Controls and Buffers contain sodium azide (NaN3) as a
preservative. NaN3 may be toxic if ingested or adsorbed by skin or eyes. NaN3 may react
with lead and copper plumbing to form highly explosive metal azides. On disposal, flush with
a large volume of water to prevent azide build-up. Please refer to decontamination
procedures as outlined by CDC or other local/national guidelines.
Do not smoke, eat or drink when manipulating the kit. Do not pipette by mouth.
All human source material used for some reagents of this kit (controls, standards e.g.) has
been tested by approved methods and found negative for HbsAg, Hepatitis C and HIV 1.
However, no test can guarantee the absence of viral agents in such material completely.
Thus handle kit controls, standards and patient samples as if capable of transmitting
infectious diseases and according to national requirements.
The kit contains material of animal origin as stated in the table of contents, handle according
to national requirements.

5.2 General directions for use

In case that the product information, including the labeling, is defective or incorrect please
contact the manufacturer or the supplier of the test kit.
Do not mix or substitute Controls, Calibrators, Conjugates or microplates from different lot
numbers. This may lead to variations in the results.
Allow all components to reach room temperature (20-32°C/68-89.6°F) before use, mix well
and follow the recommended incubation scheme for an optimum performance of the test.
Incubation: We recommend test performance at 30°C/86°F for automated systems.
Never expose components to higher temperature than 37°C/ 98.6°F.
Always pipette substrate solution with brand new tips only. Protect this reagent from light.
Never pipette conjugate with tips used with other reagents prior.
A definite clinical diagnosis should not be based on the results of the performed test
only, but should be made by the physician after all clinical and laboratory findings
have been evaluated. The diagnosis is to be verified using different diagnostic
methods.

Page 3 of 9
 Product Ref. 3222
Product Desc. Phospholipid-8Pro-A
Manual Rev. No. 003 : 2015-12-09

6 Sample Collection, Handling and Storage

Use preferentially freshly collected serum samples. Blood withdrawal must follow national
requirements. Do not use icteric, lipemic, hemolysed or bacterially contaminated samples.
Sera with particles should be cleared by low speed centrifugation (<1000 x g). Blood samples
should be collected in clean, dry and empty tubes.
After separation, the serum samples should be used during the first 8h, respectively stored
tightly closed at 2-8°C/35-46°F up to 48h, or frozen at -20°C/-4°F for longer periods

7 Assay Procedure
7.1 Preparations prior to starting
Dilute concentrated reagents:
Dilute the concentrated sample buffer 1:5 with distilled water (e.g. 20 ml plus 80 ml).
Dilute the concentrated wash buffer 1:50 with distilled water (e.g. 20 ml plus 980 ml).
To avoid mistakes we suggest to mark the cap of the different calibrators.

Samples:
Dilute serum samples 1:101 with sample buffer (1x)
e.g. 1000 µl sample buffer (1x) + 10 µl serum. Mix well !
Washing:
Prepare 20 ml of diluted wash buffer (1x) per 8 wells or 200 ml for 96 wells
e.g. 4 ml concentrate plus 196 ml distilled water.
Automated washing:
Consider excess volumes required for setting up the instrument and dead volume of robot
pipette.
Manual washing:
Discard liquid from wells by inverting the plate. Knock the microwell frame with wells
downside vigorously on clean adsorbent paper. Pipette 300 µl of diluted wash buffer into
each well, wait for 20 seconds. Repeat the whole procedure twice again.
Microplates:
Calculate the number of wells required for the test. Remove unused wells from the frame,
replace and store in the provided plastic bag, together with desiccant, seal tightly (2-8°C/35-
46°F).

Page 4 of 9
 Product Ref. 3222
Product Desc. Phospholipid-8Pro-A
Manual Rev. No. 003 : 2015-12-09

8 Qualitative Interpretation
Read the optical density of the specific calibrator (A-H) and the patient samples. Multiply the
OD of the calibrator by the parameterspecific factor, provided with the lot specific QC
certificate. For qualitative interpretation we recommend to consider sera within a range of
20% around the cut-off value as equivocal. All samples with higher ODs are considered
positive, samples with lower ODs are considered negative.
Phospholipid-8Pro-A O.D. 450/620 nm

Negative Control 0.033

Cut-off Calibrator 0.5225

Example of interpretation
We recommend pipetting cut-off calibrator in parallel for each run.
Measured: ODCut-off Calibrator (ß2-Glyco) 0.5225
Negative: OD Patient < 0.8 x OD Cut-off Parameter = 0.8 x 0.5225 =0.418

Positive: OD Patient > 1.2 x OD Cut-off Parameter = 1.2 x 0.5225 =0.627

Equivocal: 0.418 ≤ OD Patient ≤ 0.627

ID Nr. Sample OD - Calculation Interpretation

OD ß2-Glyco

1 0.99 > 0.627 --->Positive

2 0.49 ≥ 0.418 und ≤ 0.627 --->Equivocal

3 0.27 < 0.418 --->Negative

Do not use this example for interpreting patients results!

For lot specific data, see enclosed quality control leaflet. Medical laboratories might perform
an in-house Quality Control by using own controls and/or internal pooled sera, as foreseen
by EU regulations.
Each laboratory should establish its own normal range based upon its own techniques,
controls, equipment and patient population according to their own established procedures. In
case that the values of the controls do not meet the criteria the test is invalid and has to be
repeated. The following technical issues should be verified: Expiration dates of (prepared)
reagents, storage conditions, pipettes, devices, photometer, incubation conditions and
washing methods. If the items tested show aberrant values or any kind of deviation or that
the validation criteria are not met without explicable cause please contact the manufacturer
or the supplier of the test kit. For semi-quantification of the results, each patient-OD value
can be expressed by the Index-Value. The Index-Value is calculated by dividing the patient-
OD by the cut-off parameter:
OD (patient sample)
Index Value =
OD (cut-off calibrator)

Negative: Index Value < 0.8

Equivocal: 0.8≤ Index Value ≤ 1.2
Positive: Index Value >1.2

Page 7 of 9
- Diagnosi in vitro - For in vitro diagnostic use
- Pour diagnostic in vitro - Para uso diagnóstico in vitro
- Ιn Vitro Diagnostikum - In Vitro Διαγνωστικό μέσο
- Para uso Diagnóstico in vitro
¨ Numero d’ordine ¨ Cataloge number
¨ Référence Catalogue ¨ Numéro de catálogo
¨ Bestellnummer ¨ Αριθμός παραγγελίας
¨ Número de catálogo
¨ Descrizione lotto ¨ Lot
¨ Lot ¨ Lote
¨ Chargen Bezeichnung ¨ Χαρακτηρισμός παρτίδας
¨ Lote
¨ Conformità europea ¨ EC Declaration of Conformity
¨ Déclaration CE de Conformité ¨ Declaración CE de Conformidad
¨ Europäische Konformität ¨ Ευρωπαϊκή συμφωνία
¨ Déclaracão CE de Conformidade
¨ 96 determinazioni ¨ 96 tests
¨ 96 tests ¨ 96 pruebas
¨ 96 Bestimmungen ¨ 96 προσδιορισμοί
¨ 96 Testes
¨ Rispettare le istruzioni per l’uso ¨ See instructions for use
¨ Voir les instructions d‘utilisation ¨ Ver las instrucciones de uso
¨ Gebrauchsanweisung beachten ¨ Λάβετε υπόψη τις οδηγίες χρήσης
¨ Ver as instrucões de uso
¨ Da utilizzarsi entro ¨ Use by
¨ Utilise avant le ¨ Utilizar antes de
¨ Verwendbar bis ¨ Χρήση μέχρι
¨ Utilizar antes de
¨ Conservare a 2-8°C ¨ Store at 2-8°C (35-46°F)
¨ Conserver à 2-8°C ¨ Conservar a 2-8°C
¨ Lagerung bei 2-8°C ¨ Φυλάσσεται στους 2-8°C
¨ Conservar entre 2-8°C
¨ Prodotto da ¨ Manufactured by
¨ Fabriqué par ¨ Fabricado por
¨ Hergestellt von ¨ Κατασκευάζεται από
¨ Fabricado por
¨ Calibratore cut-off ¨ Cut off Calibrator
¨ Etalon Seuil ¨ Calibrador de cut-off
¨ Grenzwert Kalibrator ¨ Οριακός ορός Αντιδραστήριο βαθμονόμησης
¨ Calibrador de cut-off
¨ Controllo positivo ¨ Positive Control
¨ Contrôle Positif ¨ Control Positivo
¨ Positiv Kontrolle ¨ Θετικός ορός ελέγχου
¨ Controlo positivo
¨ Controllo negativo ¨ Negative Control
¨ Contrôle Négatif ¨ Control Negativo
¨ Negativ Kontrolle ¨ Αρνητικός ορός ελέγχου
¨ Controlo negativo
¨ Calibratore ¨ Calibrator
¨ Etalon ¨ Calibrador
¨ Kalibrator ¨ Αντιδραστήριο βαθμονόμησης
¨ Calibrador
¨ Recupero ¨ Recovery
¨ Corrélation ¨ Recuperado
¨ Wiederfindung ¨ Ανάκτηση
¨ Recuperacão
¨ Coniugato ¨ Conjugate
¨ Conjugé ¨ Conjugado
¨ Konjugat ¨ Σύζευγμα
¨ Conjugado
¨ Micropiastra rivestita ¨ Coated microtiter plate
¨ Microplaque sensibilisée ¨ Microplaca sensibilizada
¨ Beschichtete Mikrotiterplatte ¨ Επικαλυμμένη μικροπλάκα
¨ Microplaca revestida
¨ Tampone di lavaggio ¨ Wash buffer
¨ Tampon de Lavage ¨ Solución de lavado
¨ Waschpuffer ¨ Ρυθμιστικό διάλυμα πλύσης
¨ Solucão de lavagem
¨ Tampone substrato ¨ Substrate buffer
¨ Substrat ¨ Tampón sustrato
¨ Substratpuffer ¨ Ρυθμιστικό διάλυμα υποστρώματος
¨ Substrato
¨ Reagente bloccante ¨ Stop solution
¨ Solution d‘Arrêt ¨ Solución de parada
¨ Stopreagenz ¨ Αντιδραστήριο διακοπής αντίδρασης
¨ Solucão de paragem
¨ Tampone campione ¨ Sample buffer
¨ Tampon Echantillons ¨ Tampón Muestras
¨ Probenpuffer ¨ Ρυθμιστικό διάλυμα δειγμάτων
¨ Diluente de amostra